Glycoconjugates and methods

ABSTRACT

Methods for making the functionalized glycoconjugates include (a) contacting a cell with a first monosaccharide, and (b) incubating the cell under conditions whereby the cell (i) internalizes the first monosaccharide, (ii) biochemically processes the first monosaccharide into a second saccharide, (iii) conjugates the saccharide to a carrier to form a glycoconjugate, and (iv) extracellularly expresses the glycoconjugate to form an extracellular glycoconjugate comprising a selectively reactive functional group. Methods for forming products at a cell further comprise contacting the functional group of the extracellularly expressed glycoconjugate with an agent which selectively reacts with the functional group to form a product. Subject compositions include cyto-compatible monosaccharides comprising a nitrogen or ether linked functional group selectively reactive at a cell surface and compositions and cells comprising such saccharides.

This is a divisional application of the U.S. application Ser. No.08/856,865 filed on May 15, 1997, now U.S. Pat. No. 6,075,134.

The research carried out in the subject application was supported inpart by grants from the Department of Energy (Contract No.DE-AC03-76SF00098). The government may have rights in any patent issuingon this application.

FIELD OF THE INVENTION

The invention relates to functionalized saccharides and methods ofmaking and using such saccharides.

BACKGROUND OF THE INVENTION

The early diagnosis and treatment of cancer, a family of diseases thataffects one in three Americans, continues to present a major challenge(1). While primary tumors can be removed surgically, by the time theyare identified, metastatic cells may have spread throughout the bodyresulting in the lethal growth of tumors in secondary sites. As aresult, most treatment protocols involve the additional use ofchemotherapeutics, many of which are characterized by severe toxic sideeffects. These problems have motivated the search for cancer-cellspecific agents capable of facilitating the early diagnosis of tumors(i.e., before metastases have developed) and targeting the toxic effectsof drugs to cancer cells and away from normal cells. A central featureof these efforts is the identification of cell-surface antigens that arespecific to cancer cells. Several such antigens have been identifiedand, likewise, some progress has been achieved using monoclonalantibodies (mAbs) as delivery agents for drugs (“immunotoxins”) (2) anddiagnostic probes (“immunodiagnostics”) (3). Unfortunately, theheterogeneity of cancer-associated epitopes has necessitated laboriousand cumbersome mAb preparation for each of the myriad of differentcancer-associated antigens (4,5). Furthermore, the murine-based mAbs incommon usage have proven to be immunogenic in human patients (6) andattempts to “humanize” such mAbs have not yet reached maturity (7). Aparticularly refractory problem is the development of resistant cellsthat are able to mask or downregulate the targeted antigens (4,8). Thus,alternative approaches are urgently needed for selective cancer celltargeting.

The composition of cell surface carbohydrates is dramatically altered inmany epithelial and blood-derived cancers. Alterations incancer-associated cell surface carbohydrate structures can be dividedinto two broad categories: a) increased levels and inappropriateexpression of normal oligosaccharide epitopes and b) expression of novelcarbohydrate moieties that do not normally occur on healthy cells. Themajor antigen families associated with transformed cells are the sialylLewis a, sialyl Lewis x, Lewis y, A and Tn-related oligosaccharides (4).A common feature of these antigens is that each contains one or moreterminal sialic acid or fucose residues, and the Lewis antigens containboth. The overexpression of sialic acid and fucose residues is highlycorrelated with increased metastatic potential in several cancers withthe greatest impact on human health including gastric, uterineendometrial, colonic, epithelial, pancreatic, bladder, liver, lung,prostate and breast cancers as well as several types of leukemia (9).Consequently, therapeutic and diagnostic strategies that target sialicacid and/or fucose residues may have broad applicability to a variety ofcancers.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for making and usingfunctionalized glycoconjugates. For example, novel and/or unnaturalsugars are incorporated into cell associated oligosaccharides usingresident pathways of oligosaccharide biosynthesis to expand theinformational repertoire of the plasma membrane.

Disclosed methods for making glycoconjugates include (a) contacting acell with a first monosaccharide, and (b) incubating the cell underconditions whereby the cell (i) internalizes the first monosaccharide,(ii) biochemically processes the first monosaccharide into a secondmonosaccharide, (iii) conjugates the second monosaccharide to a carrierto form a glycoconjugate, and (iv) extracellularly expresses theglycoconjugate to form an extracellular glycoconjugate comprising aselectively reactive functional group. In a particular embodiments, thefirst monosaccharide comprises a chemically reactive functional groupsuch as a ketone, which is incorporated into the second monosaccharide,the glycoconjugate and the extracellular glycoconjugate, the firstfunctional group is N-linked in the first monosaccharide, and the firstmonosaccharide comprises ManLev.

The invention also includes methods for forming a wide variety ofproducts at a cell. The products may provide a label, a binding site, amodulator of cell function such as a drug or toxin, a radiativeemission, etc. These methods comprise the steps of making aglycoconjugate according to the invention and then contacting thefunctional group of the extracellularly expressed glycoconjugate with anagent which selectively reacts with the functional group to form aproduct. In a particular embodiment, the agent comprises a functionalgroup moiety, such as a hydrazide, which selectively reacts with thefunctional group of the extracellular glycoconjugate to form a covalentbond, and an effector moiety, such as a drug, which modulates a functionof a cell.

The subject compositions include cyto-compatible monosaccharidescomprising a nitrogen or ether linked functional group, such as aketone, selectively reactive at a cell surface and compositions andcells comprising such saccharides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The biosynthetic pathway for sialoglycoconjugates.

FIG. 2. Synthesis of a ricin-hydrazide conjugate.

FIG. 3. Synthesis of a bis(hydrazide) Gd³⁺chelator for ketone-directedMRI.

FIG. 4. Biosynthetic pathway for cell surface fucosides.

FIG. 5. Synthesis of FucLev, an unnatural ketone-modified fucosederivative.

FIG. 6. Biosynthetic incorporation of ketone groups into cell-surfaceassociated sialic acid.

(A) N-Levulinoyl mannosamine (ManLev) is metabolically converted to thecorresponding cell surface sialoside. (B) Cells displaying ketone groupscan be chemoselectively ligated to hydrazides under physiologicalconditions through the formation of an acyl hydrazone.

FIG. 7. Ketone expression in Jurkat, HL-60, and HeLa cells. Cellstreated with buffer alone or with ManNAc showed only a background levelof fluorescence. Cells treated with ManLev showed up to a 30-foldincrease in fluorescence above the backgrounu level, which was dependenton both biotinamidocaproyl hydrazide (Bio) and FITC-avidin (Av)treatment. Similar results were obtained in three replicate experimentsfor each cell line.

FIG. 8. Ketone groups are expressed within cell-surface sialic acids.(A) Tunicamycin inhibits ketone incorporation in a dose-dependentfashion, confirming the presence of ketones on N-linked oligosaccharides(26). Identical results were obtained in three replicate experiments.(B) Ricin binding of normal and ManLev-treated Jurkat cells with andwithout sialidase treatment. Ricin binding was quantified by stainingwith FITC-RCA₁₂₀ (Sigma) followed by analysis by flow cytometry (24).Error bars represent the standard deviation of the mean for threereplicate experiments. (C) Ketone incorporation into cell-surfacesialosides is dose dependent and saturable. Jurkat cells were incubatedwith increasing concentrations of ManNAc for 48 h, stained with Bio andAv and analyzed by flow cytometry. Data from four experiments is shown.(D) ManNAc competes with ManLev and inhibits ketone incorporation.Jurkat cells were incubated with ManLev (5 mM) and increasingconcentrations of ManNAc for 48 h, and ketone expression was quantifiedby flow cytometry. Each data point represents the average from threeexperiments and error bars represent the standard deviation of the mean.

FIG. 9. A method for engineering unique cell-surface epitopes forselective drug-delivery: cells are treated with ManLev resulting in theexpression of cell surface ketones. Reaction with biotin hydrazideresults in the display of a unique molecular target on the cell surface,and a ricin A chain-avidin conjugate selectively targets biotin-modifiedcells. with varying concentrations of ManLev.

FIG. 10. A method for engineering unique cell-surface epitopes forselective drug-delivery: toxicity of the RTA-avidin conjugate againstJurkat cells treated with varying concentrations of ManLev.

FIG. 11. Examples of functional-group bearing mannosamine derivatives.

FIG. 12. Examples of functional-group bearing fucose derivatives.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

A particular disclosed method for making glycoconjugates involves: a)contacting a cell with a first monosaccharide comprising a firstfunctional group, and b) incubating the cell under conditions wherebythe cell (i) internalizes the first monosaccharide, (ii) biochemicallyprocesses the first monosaccharide into a second monosaccharide whichcomprises a second functional group, (iii) conjugates the secondmonosaccharide to a carrier to form a glycoconjugate comprising a thirdfunctional group, and (iv) extracellularly expresses the glycoconjugateto form an extracellular glycoconjugate comprising a fourth, selectivelyreactive, functional group.

The extracellular glycoconjugate may be presented in multiple forms suchas membrane-associated, e.g. a membrane bound glycolipid orglycoprotein, associated with cell-proximate structures, e.g.extracellular matrix components or neighboring cells, or in asurrounding medium or fluid, e.g. as a secreted glycoprotein. Theselective reactivity of the fourth functional group permits selectivetargeting of the glycoconjugate as presented by the cell. For example,fourth functional groups of surface associated glycoconjugates mustprovide a reactivity that permits the selective targeting of theglycoconjugate in the context of the associated region of the cellsurface. Preferentially reactivity may be effected by a more reactivecontext, i.e. the glycoconjugate-associated fourth functional groupprovides greater accessibility, greater frequency or enhanced reactivityas compared with such functional groups present proximate to the siteof, but not associated with the glycoconjugate; or, in a preferredembodiment, the fourth functional group is unique to the region ofglycoconjugate presentation.

The selective reactivity provided by the fourth functional group maytake a variety of forms including nuclear reactivity, such as theneutron reactivity of a boron atom, and chemical reactivity, includingcovalent and non-covalent binding reactivity. In any event, the fourthfunctional group should be sufficient for the requisite selectivereactivity. A wide variety of chemical reactivities may be exploited toprovide selectivity, depending on the context of presentation. Forexample, fourth functional groups applicable to cell surface-associatedglycoconjugates include covalently reactive groups not normallyaccessible at the cell-surface, including alkenes, alkynes, dienes,thiols, phosphines and ketones. Suitable non-covalently reactive groupsinclude haptens, such as biotin, and epitopes such as dinitrophenol.

In one embodiment of the invention, the nature of the expressedglycoconjugate is a function of the first monosaccharide, the cell typeand incubation conditions. In this embodiment, the resident biochemicalpathways of the cell act to biochemically process the firstmonosaccharide into the second monosaccharide, conjugate the secondmonosaccharide to an intracellular carrier, such as anoligo/polysaccharide, lipid or protein, and extracellularly express thefinal glycoconjugate. Alternatively, the expressed glycoconjugate mayalso be a function of further manipulation. For example, the fourthfunctional group may result from modifying the third functional group asinitially expressed by the cell. For example, the third functional groupmay comprise a latent, masked or blocked group that requires apost-expression treatment, e.g. chemical cleavage or activation, inorder to generate the fourth functional group. Such treatment may beeffected by enzymes endogenous to the cell or by exogenous manipulation.Hence, the third and fourth functional groups may be the same ordifferent, depending on cellular or extracellular processing events.

As indicated, a functional group can be a masked, latent, inchoate ornascent form of another functional group. Examples of masked orprotected functional groups and their unmasked counterparts are providedin Table 1. Masking groups may be liberated in any convenient way; forexample, ketal or enols ether may be converted to corresponding ketonesby low pH facilitated hydrolysis. Alternatively, many specific enzymesare known to cleave specific protecting groups, thereby unmasking afunctional group.

TABLE 1 Examples of masking functional groups and their unmaskedcounterparts Masking group Unmasked group dialkyl ketal ketone acetalaldehyde enol ether ketone or aldehyde oxime ketone hydrazone ketonethioester thiol cobalt-complexed alkyne alkyne

In contrast, the nature of the intracellular glycoconjugate (comprisingthe third functional group) is generally solely a function of the firstmonosaccharide, the cell type and incubation conditions, i.e. the firstand second monosaccharides and the saccharide moiety incorporated intothe intracellular glycoconjugate (as well as the first, second and thirdfunctional groups) may be the same or different depending on cellularprocessing events. For example, the first monosaccharide or functionalgroup, cell and conditions may interact to form a chemically distinctsecond monosaccharide or functional group, respectively. For example,many biochemical pathways are known to interconvert monosaccharidesand/or chemically transform various functional groups. Hence,predetermined interconversions are provided by a first monosaccharide,cell and incubation condition selection.

The first monosaccharide is selected to exploit permissive biochemicalpathways of the cell to effect expression of the extracellularglycoconjugate. For example, many pathways of sialic acid biosynthesisare shown to be permissive to a wide variety of mannose and glucosederivatives. The first functional group may be incorporated into thefirst monosaccharide in a variety of ways. In preferred embodiments, thefunctional group is nitrogen or ether linked.

A wide variety of cells may be used in the disclosed methods includingeukaryotic, especially mammalian cells and prokaryotic cells. The cellsmay be in culture, e.g. immortalized or primary cultures, or in situ,e.g. resident in the organism.

The invention also provides methods for forming products at a cell.Generally, these methods involve expressing an extracellularglycoconjugate as described above wherein the expressed glycoconjugateis retained proximate to the cell; for example, by being bound tomembrane or extracellular matrix components. Then the fourth functionalgroup is contacted with an agent which selectively reacts with thefourth functional group to form a product.

A wide variety of agents may be used to generate a wide variety ofproducts. Generally, agent selection is dictated by the nature of thefourth functional group and the desired product. For example, withchemically reactive fourth functional groups, the agent provides a fifthfunctional group which selectively chemically reacts with the fourthfunctional group. For example, where the fourth functional group is aketone, suitable fifth functional groups include hydrazines,hydroxylamines, acyl hydrazides, thiosemicarbazides and beta-aminothiols. In other embodiments, the fifth functional group is aselective noncovalent binding group, such as an antibody idiotope. Inyet other embodiments, suitable agents include radioactivity such asalpha particles which selectively react with fourth functional groupscomprising radiosensitizers such as boron atoms; oxidizers such asoxygen which react with fourth functional groups comprising a surfacemetal complex, e.g to produce cytotoxic oxidative species; etc.Alternatively, a functional group on the cell surface might have uniqueproperties that do not require the addition of an external agent, e.g. aheavy metal which serves as a label for detection by electronmicroscopy. Further examples of products formed by the interaction of acell surface functional group and an agent are given in Table 2.

TABLE 2 Examples of functional groups, agents and their productsFunctional group Agent Product ketone hydrazide hydrazone dienedienophile Diels-Alder adduct thiol alpha-bromo amide thioether boronneutrons radiation biotin avidin biotin-avidin complex dinitrophenol(DNP) anti-DNP antibodies DNP-antibody complex Fluorescein UV lightgreen light iron complex oxygen peroxy radicals

Frequently, the agent comprises an activator moiety which provides adesired activity at the cell. A wide variety of activator moieties maybe used, including moieties which alter the physiology of the cell orsurrounding cells, label the cell, sensitize the cell to environmentalstimuli, alter the susceptibility of the cell to pathogens or genetictransfection, etc. Exemplary activator moieties include toxins, drugs,detectable labels, genetic vectors, molecular receptors, and chelators.

The invention provides a wide variety of compositions useful in thedisclosed methods, including compositions comprising cyto-compatiblemonosaccharides comprising a functional group, preferably a nitrogen orether linked functional group, which group is selectively reactive at acell surface. Exemplary functional groups of such compounds includealkynes, dienes, thiols, phosphines, boron and, especially, ketones.Exemplary mannose and fucose derivatives are shown in FIGS. 11 and 12,respectively. The term substituted or unsubstituted alkyl is intended toencompass alkoxy, cycloalkyl, heteroalkyl, etc. Similarly, the termsubstituted or unsubstituted aryl is intended to encompass aryloxy,arylalkyl (including arylalkoxy, etc.), heteroaryl, arylalkynyl, etc.;the term substituted or unsubstituted alkenyl is intended to analogouslyencompass cycloalkenyl, heteroalkenyl, etc.; etc. Analogous derivativesare made with other monosaccharides having permissive pathways ofbioincorporation. Such monosaccharides are readily identified inconvenient cell and protein-based screens, such as described below. Forexample, functionalized monosaccharides incorporated into cell surfaceglycoconjugates can be detected using fluorescent labels bearing acomplementary reactive functional group (i.e., agent). A cell-basedassay suitable for mechanized high-throughput optical readings involvesdetecting ketone-bearing monosaccharides on cell surfaces by reactionwith biotin hydrazide, followed by incubation with FITC-labeled avidinand the quantitating the presence of the fluorescent marker on the cellsurface by automated flow cytometry. A convenient protein-based screeninvolves isolating the glycocojugate, e.g. gel blots, affinityimnmobilization, etc, and detecting with the complementary reactiveprobe, e.g. detone-bearing glycoconjugates detected with biotinhydrazide, followed by incubation with avidin-alkaline phosphatase oravidin-horseradish peroxidase. Alternatively, monosaccharides bearingunusual functional groups can also be detected by hydrolysis of theglycoconjugate followed by automated HPLC analysis of themonosaccharides.

For use in methods applied to cells in situ, the compositions frequentlyfurther comprise a physiologically acceptable excipient and/or otherpharmaceutically active agent to form pharmaceutically acceptablecompositions. Hence, the invention provides administratively convenientformulations of the compositions including dosage units which may beincorporated into a variety of containers. For in situ administration,the compositions are provided in any convenient way, such as oral,parenteral or topical routes. Generally the compounds are administeredin dosages ranging from about 2 mg to up to about 2,000 mg per day,although variations will necessarily occur depending on the methodapplication or target, the host and the route of administration.Preferred dosages are administered orally in the range of about 0.05mg/kg to about 20 mg/kg, more preferably in the range of about 0.05mg/kg to about 2 mg/kg, most preferably 0.05 to about 0.2 mg/kg of bodyweight per day.

EXPERIMENTAL PROCEDURES

The biosynthesis of N-acetylneuraminic acid (NeuAc, the most abundantmember of the sialic acid family) glycoconjugates is shown in FIG. 1.N-Acetylmannosamine (ManNAc) is phosphorylated and then condensed withphosphoenol pyruvate (PEP) to form NeuAc-9-phosphate in a reactioncatalyzed by the enzyme NeuAc-9-phosphate synthetase (10). Afterdephosphorylation, NeuAc is converted to CMP-NeuAc by CMP-sialic acidsynthetase. CMP-NeuAc is transported into the appropriate cellularcompartments, and the NeuAc residue is transferred by asialyltransferase onto the terminus of oligosaccharides attached toproteins or lipids.

We hypothesized that such saccharide biochemical pathways of the cellmight be appropriated for the presentation of unique functional groupson cell surfaces. First, experiments have been reported suggesting thatindividual, isolated enzymes of the sialic acid biosynthetic pathway maybe permissive for substrate variants in vitro (11-15). Furthermore,conservative changes within a natural functional group (the N-acetylgroup of ManNAc extended to N-propanoyl, N-butanoyl or N-pentanoyl) maybe also be permitted by the same pathway in cell culture and in vivo(16,17). While our hypothesized permissive introduction of entirely newfunctional groups was without precedent—the biosynthetic machinery for.the other major biopolymers (i.e., proteins and nucleic acids) is quiterestrictive—we anticipated that, if possible, such oligosaccharideswould prove remarkably versatile hosts for biosynthetic labels,effectors and probes in vivo.

We initially chose the ketone for cell surface display based on twoconsiderations. First, the ketone is chemically unique to the cellsurface since none of the naturally occurring amino acids,glycoconjugates or lipids possess a ketone group. Second, the ketone canbe chemoselectively ligated with hydrazides to form the correspondingacyl hydrazones under physiological conditions, and shows no appreciablereactivity with the functional groups found in biomolecules. Theseproperties of the ketone group have been widely exploited for thechemoselective ligation of proteins and small molecules (18,19).Furthermore, hydrazone formation between small molecule drugs has beenaccomplished in whole animals and human subjects, setting the precedentfor the application of our cell surface targeting approach toanti-cancer therapy (20). Although ketones and hydrazides react readilyat normal physiological pH (pH=7.3-7.6), the reaction rate is enhancedup to 10-fold at lower pHs (pH=5). Because the extracellular pH in manytumors is slightly lower (ranging as low as 5.6) than in normal tissue(21), hydrazide-conjugated agents have an additional level ofselectivity for tumor environments over normal tissue.

The prevalence of sialic acid on cancer-associated glycoproteins andglycolipids makes this residue an ideal vehicle for the presentation ofketone groups capable of directing hydrazide-conjugated toxins or probesto cancer cells. We initially demonstrated the feasibility of thisapproach with the plant-derived toxin ricin, which has been widelyexplored for use in cancer therapy. Ricin is composed of two chains, Aand B, linked together by a disulfide bond. The B chain binds tocell-surface galactose residues, thereby delivering the toxic A chain tothe cell surface which is followed by internalization via constitutiveendocytotic processes. As demonstrated in the Examples below, weconstructed a modified ricin derivative in which hydrazide groups areattached to the surface of the A chain via disulfide linkers, replacingthe B chain altogether and providing a targeting mechanism toketone-coated cells (FIG. 2). Ricin A chain was treated withiminothiolane to convert two surface-accessible lysine residues to thecorresponding thiol derivatives, affording a total of three accessiblethiols for further conjugation. Further reaction with PDPH installed thedesired hydrazide groups.

The toxicity of ricin-hydrazide is readily evaluated with a variety oftransformed cell types in the presence and absence of ketone expression.For example, with Jurkat lymphoma, the concentrations of ricin-hydraziderequired for 50% cell death (LD₅₀) is evaluated by treatment of cellcultures with the toxin for 2 hours, followed by removal of the toxinfrom the suspension and incubation of the cells for an additional 24 h.Live cells are counted using a standard haemocytometer-based assay andcomparisons made between ketone-coated and unmodified cells, usingnative ricin A chain as a control for background toxicity. With theoptimal parameters established, a panel of transformed cell types arecompared with their non-transformed counterparts with respect to ketoneincorporation and LD₅₀ of ricin-hydrazide. In addition, our drugdelivery efforts are extended to other toxins requiring a cell-surfacetarget for optimal activity,. e.g. abrin toxin and diptheria toxin, andhydrazide-ketone ligation is used to deliver small molecule drugs tocells via a cleavable disulfide linker.

The availability of cancer-specific magnetic resonance imaging (MRI)contrast reagents has revolutionized our ability to diagnose lesions attheir earliest stages. The most commonly used contrast reagents arebased on Gd³⁺in the form of a protein conjugate (3). We have designed ahydrazide-conjugated Gd³⁺reagent (FIG. 3) capable of imagingketone-expressing cells selectively. The synthesis of this complexinvolves the reaction of the commercially available DTPAA with adipicacid dihydrazide to form the bis-hydrazide product which chelates Gd³⁺inan essentially irreversible manner. This derivative reacts specificallywith ketones expressed on highly sialylated tumor cells. Again, sincethe hydrazone formation reaction is accelerated in acidic environments,further selectivity for tumor cells is achieved.

Initial demonstrations are conducted on cultured lymphoma cells thathave been treated with ManLev to induce ketone expression. We use Eu³⁺asa Gd³⁺mimic for initial experiments since Eu³⁺is highly fluorescent(emission max=590 nm) and is therefore amenable to flow cytometryanalysis. The cells are stained with the bis-hydrazide chelator loadedwith Eu³⁺cations, then washed and analyzed by flow cytometry. Theoptimal parameters (i.e., concentration of chelator and time ofincubation) for selective cell-surface staining are determined,particularly, the staining levels of mixed populations of normal andcancerous lymphocytes after parallel treatment with ManLev. In a twocolor flow cytometry experiment, we correlate Eu³⁺staining with thelevel of staining of specific tumor or normal cell markers, showing thatcancerous lymphocytes are stained at significantly higher levels thattheir normal counterparts. This correlation demonstrates the relativespecificity of our hydrazide-based contrast reagent.

With the optimal staining parameters established in vitro, wedemonstrate ManLev uptake and Gd³⁺staining in mice and in rabbits. Mice(balb/c) are injected IV with various doses of ManLev in 8 boluses overa 2-day period using a protocol for the uptake and cellularincorporation of mannosamine derivatives (22). The mice are then beinjected with the Gd³⁺-loaded bis(hydrazide) chelator and after a 6 hourperiod the mice are anesthetized and subjected to whole animal MRI.

To determine the selectivity of ManLev uptake and metabolism in micecarrying surgically implanted tumors, the Gd³⁺-loaded bis(hydrazide)chelator is used as a marker for ketone presentation in vivo, andcorrelations made among tumor type and staining intensity. Organs thatstain at high levels are identified and membrane glycoproteins fromthese tissues isolated and biochemically characterized to confirm thepresence of high levels of ketone-modified sialic acids. In addition,the staining levels of normal organs are compared to the correspondingcancerous tissue to evaluate the selectivity of Gd³⁺localization.

In addition to high levels of sialic acid, many cancer cells expresselevated levels of cell surface fucose residues. There are two pathwaysfor the biosynthesis of fucosides: one begins with mannose and the otherwith free fucose. The latter pathway is advantageous in that since itinvolves fewer transformations and, therefore, fewer potentialbiosynthetic bottlenecks. Fucose is taken up from the extracellularmilieu and converted to the activated form, GDP-fucose, by a GDP-fucosesynthetase (FIG. 4). The fucose residue is then transferred onto theterminus of an oligosaccharide chain by a fucosyltransferase within theGolgi compartment. A recent in vitro study reported that a humanfucosyltransferase may be permissive for substituents attached at C-6,which is a simple methyl group in the native sugar (23). Hence,.whypothesized that the C-6 position might also be a promising site forderivitization of fucose with a ketone functionality. 6-N-levulinamidofucose (FucLev), which possesses a ketone groups, from L-galactose issynthesized as shown in FIG. 5. L-Galactose is protected as the 1,2-3,4di-O-ispropylidene derivative. The 6-OH is converted first to themesylate and then to the corresponding azide. Reduction followed bydeprotection affords 6-amino fucose. Finally, acylation with levulinicacid provides the desired ketone-modified fucose derivative. The uptakeand metabolism of this compound is. evaluated in Jurkat lymphomas andHL60 cells (a human neutrophil cell line), both of which express highlevels of fucose on cell surface glycoconjugates. The expression ofketones is assayed with biotin hydrazide followed by flow cytometryanalysis as described for sialic acid-associated ketones, and the sametargeting strategies using ricin-hydrazide and the Gd³⁺-loadedbis(hydrazide) chelator are applied.

Synergistic tumor cell targeting is effected by incorporating twoorthogonal unnatural functional groups into sialic acid and fucoseresidues in the same cancer cell. Cells with high levels of both sialicacid and fucose are more susceptible to agents capable of binding bothfunctional groups.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

We synthesized N-levulinoyl mannosamine (ManTev) (FIG. 6A), which hasthe ketone functionality at the position normally occupied by theN-acetyl group in the natural substrate ManNAc. We selected three humancell lines, Jurkat (T cell-derived), HL-60 (neutrophil-derived) and HeLa(cervical epithelial carcinoma), to test the biosynthetic conversion ofManLev to the corresponding cell-surface associated, unnatural sialicacid (FIG. 6A). Cells were treated with ManLev and the presence ofketone groups on the cell surface was determined by the chemoselectiveligation of a hydrazide-based probe, biotinamidocaproyl hydrazide (FIG.6B). Note that in this example, cell surface ketones were conjugated tobiotinamidocaproyl hydrazide to provide a tag for subsequent detectionwith FITC-avidin; however, any hydrazide-derivatized molecule can beused selectively remodel the surface of ketone-expressing cells. Thecells were analyzed by flow cytometry after staining with FITC-avidin(24). The Jurkat, HL-60, and HeLa cells treated with ManLev showed alarge increase in fluorescence intensity compared to cells treated withbuffer or the natural derivative ManNAc (FIG. 7). ManLev-treated cellsthat were stained with FITC-avidin alone, without priorbiotinamidocaproyl hydrazide treatment, showed only a background levelof fluorescence. These results indicate that ManLev-treated cellsexpress cell surface-associated ketone groups and can bechemoselectively decorated with hydrazide conjugates, even in thepresence of serum.

We performed a series of experiments to demonstrate that the ketonegroups are displayed on the cell surface in the form of modifiedsialoglycoconjugates. Jurkat cells were treated with tunicamycin, aninhibitor of N-linked protein glycosylation, prior to incubation withManLev (25,26). We anticipated a dramatic reduction in ketone expressionon the basis of the observation that most mature (and thereforesialylated) oligosaccharides on Jurkat cells are found on N-linkedrather than O-linked glycoproteins (27). Indeed, ketone expressionresulting from ManLev treatment was inhibited by tunicamycin in adose-dependent fashion (FIG. 8A), indicating that the ketone groups arepresented on oligosaccharides and are not nonspecifically associatedwith cell-surface components. In contrast, ketone expression in HL-60and HeLa cells was unaffected by tunicamycin, but was instead blocked byα-benzyl N-acetylgalactosamine, an inhibitor of O-linked glycosylation,consistent with the high expression of mucin-like molecules on myeloid-and epithelial-derived cell lines.

Although we predicted that ManLev would be converted into thecorresponding sialoside, we addressed the possibility that ketoneexpression resulted from conversion of ManLev to N-levulinoylglucosamine (GlcLev) by the enzyme that interconverts ManNAc and GlcNAc.In that GlcNAc is incorporated into most glycoproteins, GlcLev wouldhave many avenues for cell surface expression. Synthetic GlcLev wasincubated with Jurkat cells, and flow cytometry analysis revealed onlybackground fluorescence, indicating that unnatural sialosides are themajor biosynthetic products of ManLev.

Since the commercially available sialidases were found to be inactiveagainst N-levulinoyl sialosides, in accordance with their known reducedactivity against sialosides with other unnatural N-acyl groups (28),abrogating the fluorescence signal by treatment with sialidase enzymesto show cell-surface expression of ketone-bearing sialic acids was notpractical. We therefore evaluated the effects of ManLev treatment on theamount of normal sialic acid on Jurkat cells, expecting a reduction.Indeed, the amount of sialic acid released from ManLev-treated cells bysialidase digestion, as quantified by high-pH anion exchangechromatography (HPAEC), was found to be approximately 10-fold lower thanthat released from ManNAc-treated cells (29).

Two possible explanations for the observed reduction in normal sialicacid on ManLev-treated cells are (i) normal sialic acid is replaced withthe unnatural sialic acid during incubation with ManLev, or (ii) thebiosynthesis of all sialosides is suppressed during incubation withManLev. Inhibition of sialoside biosynthesis would cause an increase inexposed terminal galactose residues, the penultimate residue in themajority of sialoglycoconjugates. We therefore examined the effect ofManLev treatment on the binding of the galactose-specific lectin ricinto Jurkat cells (30). Ricin binding to ManLev-treated and untreatedcells was found to be identical (FIG. 8B), indicating thatManLev-treatment does not inhibit sialoside biosynthesis. Sialidasetreatment of normal Jurkat cells increased ricin binding over backgroundlevels as expected (31). When ManLev-treated cells were digested withsialidase, however, a much smaller increase in ricin binding wasobserved. This finding indicates the substitution of normal sialic acidswith unnatural sialic acids refractory to enzymatic cleavage inManLev-treated cells.

We have also determined the quantitative and physiological limits to thecell surface expression of reactive functional groups. Ketone expressionis dose dependent and saturable in ManLev-treated Jurkat cells (FIG.8C). At saturation, we calculated the number of ketones accessible tochemoselective ligation and flow cytometry analysis to be approximately1.8×10⁶ per cell (32). No effects on cell morphology or the rate of cellgrowth were observed during prolonged (up to 6 weeks) treatment withManLev. We therefore conclude that even at maximal levels, the presenceof sialic acid-associated ketones does not grossly alter normal cellularfunctions. Additionally, we demonstrated that ketone expression wasinhibited by the addition of ManNAc to ManLev-treated cells, confirmingthat both substrates compete in the same biosynthetic pathway (FIG. 8D).

Our ability to manipulate the chemical reactivity of cell surfaces usingbiosynthetic processes opens the door to a myriad of applications. Forexample, cell surfaces can be engineered to present unique epitopes forthe selective targeting of drugs, radionucleotides or imaging reagents,an alternative to well known immunotargeting strategies. Sialic acidresidues are overexpressed on a number of human cancers (33), offeringthe expression of unnatural, reactive sialic acids as a possiblemechanism to differentiate cancer cells from normal cells in a newtargeting strategy.

We have demonstrated the viability of this approach by decorating cancercell surfaces with biotin hydrazide, providing a unique cell surfaceepitope for the delivery of avidin-conjugated toxins (FIG. 9A). We chosethe ricin toxin A chain (RTA), a potent inhibitor of protein synthesis,as a model toxin on the basis of precedents in the field of immunotoxinresearch (34,35) and, accordingly, we prepared a disulfide-linkedRTA-avidin conjugate using previously described methods (36,37). Thedisulfide linkage is commonly used in the preparation ofimmunoconjugates since it provides a mechanism for toxin release oncethe cell surface-bound conjugate enters the reducing environment of thecell's interior (38,39).

The selective toxicity of the RTA-avidin conjugate was evaluated usingJurkat cells treated with varying concentrations of ManLev and thendecorated with the targeting epitope, biotin hydrazide (FIG. 9B; 40).The toxicity of the conjugate was found to be dependent on the level ofketone expression: cells expressing high levels of ketones (>700,000ketones/cell as estimated by flow cytometry analysis) were sensitive tothe conjugate with LD₅₀'s in the 1-10 nM range. In contrast, theconjugate showed no toxicity against cells expressing low numbers ofketones (<50,000 ketones/cell). These results indicate that cellsurfaces can be metabolically engineered to support selective drugdelivery, and that the sensitivity of target cells can be controlled bymodulating the expression level of the unique targeting epitope.

Other variations of this strategy are provided, such as the directtargeting of cell surface ketones with hydrazide-conjugated drugs orprobes and the use of other mutually reactive organic functional grouppairs. It should be noted that the chemoselective formation of hydrazonelinkages among small molecule drugs has been accomplished in wholeanimals and human subjects. Other applications of cell surfaceremodeling include engineering new determinants for immunologicalrecognition, tissue-specific cell trafficking, and cell adhesion tosynthetic substrates.

Parenthetical References

1. Scientific American September, 1996.

2. J. M. Lord, L. M. Roberts, J. D. Robertus, FASEB J. 8, 201 (1994).

3. K. P. Aicher, et al., Cancer Res. 50, 7376 (1990).

4. (a) S. Sell, Human Path. 21, 1003 (1990). (b) W. J. Snell, et al.,Cell 85, 629- (1996).

5. T. H. Brununendorf, et al., Cancer Res. 54, 4162 (1994).

6. N. S. Courtenay-Luck, et al. Cancer Res. 46, 6489 (1986).

7. J. R. Couto, et al., Cancer Res. 55, 5973s (1995).

8. R. Chignola, et al., Int. J Cancer 61, 535 (1995).

9. T. Dohi, et al., Cancer 73, 1552 (1994).

10. L. Warren, Bound carbohydrates in nature (Cambridge Univ. Press, NewYork, 1994).

11. R. E. Kosa, et al., Biochem. Biophys. Res. Commun. 190, 914 (1993).

12. W. Fitz, C.-H. Wong, J Org. Chem. 59, 8279 (1994).

13. S. L. Shames, et al., Glycobiology 1, 187 (1991).

14. M. A. Sparks, et al., Tetrahedron 49, 1 (1993).

15. C.-H. Lin, et al., J. Am. Chem. Soc. 114, 10136 (1992).

16. H. Kayser, et al., J Biol. Chem. 267, 16934 (1992).

17. 0. T. Keppler, et al., J BioL Chem. 270, 1308 (1995).

18. K. Rose, J Am. Chem. Soc. 116, 30 (1994).

19. H. F. Gaertner, et al., Bioconjugate Chem. 3, 262 (1992).

20. D. Rideout, et al., Biopolymers 29, 247 (1990).

21. J. R. Griffiths Br. J Cancer 64, 425 (1991).

22. H. Kayser, C. Ats, J. Lehmann, W. Reutter, Experientia 49, 885(1993).

23. G. Srivastava, et al., J Biol. Chem. 267,22356 (1992).

24. Cultures of 2×10⁶ cells were grown in media (DME) containing ManLev(5 mM), ManNAc or no sugar for 48 hours. Cells were then washed twicewith biotin staining buffer (0.1% newborn calf serum (NCS) in phosphatebuffered saline (PBS) pH=6.5) and resuspended at a density of 10⁷cells/mL. Aliquots of 2×10⁶ cells were suspended in 1.4 mL of biotinstaining buffer and 400 μL of biotinamidocaproyl hydrazide (5 mMsolution in PBS) or 400 μL of buffer. After 2 hours at rt, the cellswere pelleted and washed twice with ice cold avidin staining buffer(0.1% NaN₃, 0.1% NCS in PBS pH=7.4). The cells were then suspended in100 μL of FITC-avidin staining solution (5.6 μg/mL of FITC-avidin inavidin staining buffer). After a 10 minute incubation in the dark at 0°C., the cells were diluted with 2 mL of cold avidin staining buffer andwashed twice. The cells were resuspended in 400 μL of avidin stainingbuffer and subjected to flow cytometry analysis.

25. F. M. Ausubel, Ed., Inhibition of N-Linked glycosylation, vol. 2(John Wiley & Sons, New York, 1994).

26. Cultures of 2×10⁶ Jurkat cells were grown in 9 mL of mediacontaining varying amounts of a 1 mg/mL solution of tunicamycin in EtOH(4.5, 7.0, 10.0 μL). After 24 hours, 1 mL of a 50 mM solution of ManLevwas added. After an additional 48 hours, the cells were washed twicewith biotin buffer (0.1% NCS in PBS pH=6.5) and resuspended at a densityof 10⁷ cells/mL.

27. V. Piller, F. Piller, M. Fukuda, J Biol. Chem. 265, 9264 (1990).

28. R. Drzeniek, Histochem. J 5, 271 (1973).

29 B. Potvin, T. S. Raju, P. Stanley, J. BioL Chem. 270, 30415

30. G. L. Nicolson, J. Blaustein, M. E. Etzler, Biochemistry 13, 196(1974).

31. Jurkat cells were grown in the presence and absence of ManLev (20mM) for 72 hours. Cells (2×10⁵ per sample) were washed with phosphatebuffered saline (PBS) (pH 6.5), centrifuged and resuspended in 0.9 mL ofsialidase buffer (20 mM HEPES, 140 mM NaCl, pH 6.8). Sialidase(Clostridium perfringes, 100 mU in 100 μL) or sialidase buffer (100 μL)was added to the cells and they were incubated at 37° C. for 30 minutes.The cells were centrifuged, washed with PBS (pH=7.4) and resuspended in0.5 mL of 25 nM FITC labeled Ricinus communis agglutinin (FITC-RCA₁₂₀Sigma). Cells were incubated on ice with the FITC-RCA,₁₂₀ for 15 minuteswashed twice with PBS (pH=7.4) and analyzed by flow cytometry.

32. The relationship between fluorescence intensity observed by flowcytometry analysis and the number of fluorescent molecules per cell wasdetermined using biotinylated polystyrene beads (Spherotech) with apre-determined number of biotin molecules per bead and with a similardiameter to Jurkat cells.

33. S. Sell, Human Pathology, 21, 1003 (1990) and references therein.

34. L. Barbieri, M. G. Battelli, F. Stirpe, Biochem. Biophys. Acta,1154, 237 (1993).

35. J. M. Lord, L. M. Roberts, J. D. Robertus, FASEB J., 8, 201 (1994).

36. A. J. Cumber, et al., Methods Enzymol., 112, 207 (1985).

37. The amino groups of lysine residues on the ricin A chain weremodified to present sulffiydryl groups by treatment with 2-iminothiolane(2-IT) as follows. Ricin toxin A chain (RTA) (1.0 mg, Sigma) wasexchanged into PBS (pH 8.5) by Sephadex G-25 gel filtration andconcentrated to a final volume of 3 mL. A 120 μL aliquot of a solutionof 2-iminothiolane (2-IT) (0.5 M in 0.8 M boric acid, pH 8.0, 50 mMdithiothreitol (DTT)) was added to the RTA solution and incubated for 2hours at room temperature. Excess 2-IT and DTT were removed by SephadexG-25 gel filtration in PBS buffer (pH 7.4). The volume of the RTA-2-ITadduct was reduced to 0.5 mL using a centricon-10 concentrator. Theamino groups of lysine residues on avidin were modified to presentpyridyldithio groups by reaction with N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) (Pierce) as follows. A solution ofegg-white avidin (Sigma, 2 mg) and SPDP (4 μL of a 250 mM solution indimethylsulfoxide) was incubated at room temperature for 1 hour. ExcessSPDP was removed by Sephadex G-25 gel filtration and the modified avidinwas collected into PBS buffer (pH 7.4). RTA-IT was added to thissolution in a 1:1 molar ratio and incubated at room temperature for 18hours. The 1:1 RTA-avidin conjugate was purified from the reaction bySephadex G-150 gel filtration and characterized by non-reducing andreducing SDS-PAGE.

38. H. T. Wright and J. D. Robertus, Archives Biochem. Biophys., 256,280(1987).

39. S. Ramakrishnan and L. L. Houston, Cancer Res., 44,201(1984). Jurkatcells were grown with (or without) ManLev and labeled withbiotin-hydrazide in PBS buffer containing 5% NCS as describedpreviously. The cells were washed and resuspended in PBS (pH 7.4, 0.1%NCS) at a density of 5×10⁵ cells/mL. Cells (100 μL) were added to 100 μLof RTA-avidin diluted to various concentrations in PBS with 0.1% NCS.The cells were incubated for 15 minutes at room temperature, and thenwashed twice to remove excess RTA-avidin. The cells were resuspended in1.0 mL of media (RPMI-1640 with 5% NCS) and incubated for 3 days. Thenumbers of living and dead cells were determined by trypan blue stainingfollowed by counting a minimum of 500 cells/sample under a lightmicroscope. Similar results were obtained in three separate experiments.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A compound of a formula selected from the groupconsisting of

wherein R₁ is a substituted or unsubstituted alkylene, alkenylene,alkynylene or arylene and R₂ is a substituted or unsubstituted alkyl,alkenyl, alkynyl or aryl wherein n is
 2. 2. The compound of claim 1depicted by the formula

wherein R₁ is alkylene, alkenylene, alkynylene or arylene; and whereinR₂ is an alkyl.
 3. The compound of claim 1 depicted by the formula

wherein R₁ is alkylene, alkenylene, alkynylene or arylene and wherein R₂is alkyl, alkenyl, alkynyl or aryl.
 4. The compound of claim 1 depictedby the formula

wherein R₁ is alkylene, alkenylene, alkynylene or arylene, wherein R₂ isalkyl, alkenyl, alkynyl or aryl and n is
 2. 5. The compound of claim 1depicted by the formula

wherein R₁ is alkylene, alkenylene, alkynylene or arylene.
 6. Thecompound of claim 1 depicted by the formula

wherein R₁ is a divalent alkylene, alkenylene, alkynylene or arylene. 7.A compound of a formula selected from the group consisting of

wherein R₁ a substituted or unsubstituted alkylene, alkenylene,alkynylene or arylene and R₂ is a substituted or unsubstituted alkyl,alkenyl, alkynyl or aryl wherein n is
 2. 8. A compound of the formula;


9. A compound of the formula:

wherein R₁ is a substituted or unsubstituted alkyl, alkenyl, alkynyl oraryl, R₂ is a substituted alkylene, alkenylene, alkynylene or aryleneand R₃ and R₄ are independently selected from O, S or H.
 10. Thecompound of claim 9 depicted by the formula


11. The compound of claim 9 depicted by the formula


12. The compound of claim 9 depicted by the formula

namely, 6-N-levulinamido fucose (FucLev).
 13. The compound of claim 9depicted by the formula


14. The compound of claim 9 depicted by the formula


15. The compound of claim 9 depicted by the formula